L-Glutamate has been known as an excitatory neurotransmitter in the mammalian central nervous system, which not only induces rapid neurotransmission between synapses, but also participates in high-order and complex physiological processes such as memory and/or learning. Excitatory neurotransmission between synapses begins with release of glutamate from presynapses and ends with rapid uptake of glutamate from synaptic clefts by the action of high-affinity glutamate transporters present in nerve endings and glial cells (Attwell, D. and Nicholls, D., TIPS 68-74, 1991).
In certain genetic neurodegenerative diseases, a decrease in sodium-dependent glutamate uptake activity has been reported in the brains of some patients (Rothstein, J. D. et al., N. Eng. J. Med 326, 1464-1468, 1992). This directs attention to the expression and inhibition of glutamate transporter functions in connection with these diseases.
Under these circumstances, there is a demand for the development of transporter-specific inhibitors, particularly those acting as blockers, in order to elucidate transport mechanisms of glutamate transporters and to study relations between glutamate transporters and neurodegenerative diseases such as neuropathies including epilepsy, Huntington's disease, amyotrophic lateral sclerosis (ALS) and Alzheimer's disease.
The inventor of the present invention has already reported that β-hydroxyaspartic acid derivatives having a substituent at the β-position exhibit uptake inhibitory effect against all of the subtypes EAAT1 to EAAT5 (Lebrun, B. et al., J. Biol. Chem. 272, 20336-20339, 1997; Shimamoto, K. et al., Mol. Pharmacol. 53, 195-201, 1998; Shigeri, Y. et al., J. Neurochem, 79, 1207-1216, 2001). The inventor has found that especially compounds having a bulky substituent at the β-position act as blockers in all the subtypes and inhibit not only glutamate uptake, but also heteroexchange-induced glutamate leakage or sodium ion influx (Chatton, J-Y. et al., Brain Res. 893, 46-52, 2001). In particular, L-threo-β-benzyloxyaspartic acid (L-TBOA) shown in Formula (2) has a potent blocker effect, but has a lower affinity for glutamate receptors than existing inhibitors. For this reason, this compound has come to be used as a standard substance in glutamate transporter studies.

The inventor has further found that compounds having an amino substituent at the meta-position of the benzyl group in L-TBOA show potent activity, and also have reported that especially benzoylamide compounds substituted at the para-position have high affinity (Shimamoto, K. et al., Mol. Pharmacol. 65, 1008-1015, 2004). Above all, the compound having a trifluoromethylbenzoyl group (TFB-TBOA) shown in Formula (3) was found to have an IC50 value for uptake inhibitory activity being in the order of nM.

On the other hand, protein purification is necessary to elucidate the three-dimensional structure of glutamate transporters to thereby clarify their substrate transport mechanisms and/or substrate binding sites. Affinity column chromatograph is an effective means for protein purification. Some attempts have been made to use antibodies for protein purification, but such attempts were disadvantageous in that proteins would lose their inherent functions during elution because of too strong a binding between antibody and protein. If a blocker can be used as a column ligand, elution under mild conditions will be permitted. For this reason, there has been a demand for a blocker that has a substituent capable of binding to an affinity column while showing potent L-glutamate uptake inhibitory activity. Among the above substituted TBOAs previously developed, those having an amino acid or biotin as a substituent can be expected to act as affinity column ligands, but their affinity was not sufficient (in the order of μM). Moreover, additional means have also been required to detect purified proteins.